Vinblastine oxazolidinedione disulfides and related compounds

ABSTRACT

Thioalkyl derivatives of vinblastine 3-spiro-5&#34;-oxazolidine-2&#34;,4&#34;-dione, mitotic inhibitors.

BACKGROUND OF THE INVENTION

The alkaloids obtainable from Vinca rosea (Catharanthus rosea) haveconstituted a most productive source for drugs which adversely affectthe growth of experimental malignancies in mammals. Initially, only someof the alkaloids obtainable from the leaves of the plant by extractionand purifiable by chromatography were found to be active. It has beenfound that all these active anti-neoplastic Vinca alkaloids obtaineddirectly from the plant are dimeric indole-dihydroindole alkaloids whichcan be represented by the formula: ##STR1##

In the above formula where R¹ is acetoxy, R² is methyl, R³ is hydroxyl,R⁴ is ethyl and R⁵ is H, vinblastine (vincaleucoblastine, VLB) isrepresented; where R¹ is acetoxy, R² is formyl, R³ is hydroxyl, R⁴ isethyl and R⁵ is H, vincristine (leurocristine) is represented; where R¹is acetoxy, R² is methyl, R³ is ethyl, R⁴ is hydroxy, and R⁵ is H,leurosidine is represented; where R¹ is acetoxy, R² is methyl or formyl,R³ is ethyl and R⁴ and R⁵ taken together form an α-epoxide ring,leurosine and leuroformine, respectively are represented. Literaturereferences to the above alkaloids are as follows: leurosine (U.S. Pat.No. 3,370,057), VLB (U.S. Pat. No. 3,097,137), leuroformine (BelgianPat. No. 811,110); leurosidine (vinrosidine) and vincristine (both inU.S. Pat. No. 3,205,220).

Two of the above alkaloids, vinblastine and vincristine, are nowmarketed for the treatment of malignancies, particularly the leukemiasand related diseases, in humans. The two marketed alkaloids arecustomarily administered by the iv route. Two others, leurosidine andleuroformine, have been on clinical trial in the United States or inEurope.

Chemical modification of the Vinca alkaloids started slowly for severalreasons. In the first place, the molecular structures involved areextremely complex, and chemical reactions which modify one specificfunctional group of the molecule without affecting other groups havebeen difficult to develop. Secondly, dimeric alkaloids lacking desirablechemotherapeutic properties have been recovered or produced from Vincarosea extracts, and a determination of their structures has led to theconclusion that these inactive compounds are closely relatedstructurally to, or even isomeric with, an active alkaloid.

One of the more recent, and more successful, modifications of the basicindole-dihydroindole structure has been the preparation of C-3carboxamide and carboxhydrazide derivatives. Many of these carboxamidesare active anti-tumor agents (see U.S. Pat. No. 4,166,810, Barnett etal., J. Med. Chem., 21, 88 (1978), id, 22, 391 (1979). In particular,4-desacetyl VLB C-3 carboxamide (vindesine) is very active and iscurrently on clinical trial in humans.

A related approach is described in United States patent 4,096,148wherein the preparation of various unsubstituted or substituted3-spiro-5"-oxazolidine-2",4"-dione derivatives of VLB, desacetyl VLB and4-desacetyl vincristine are described among other compounds. TypicalVinca oxazolidinedione derivatives found in the above patent can beportrayed by the following formula ##STR2## wherein R is H, C₁ -C₄alkyl, C₃ -C₄ alkenyl, CH₂ --CHX--CH₃

or CH₂ --CH₂ X;

wherein

X is Br or Cl;

R¹ is OH or ##STR3## R² is CH₃ or CHO; one of R³ and R⁴, is OH or H andthe other is C₂ H₅.

According to U.S. Pat. No. 4,096,148, these oxazolidinediones areprepared by reaction of, for example, vinblastine with an isocyanateRNCO (where R is other than H). One of the compounds thus prepared is3"-(2-chloroethyl)-3-spiro-5"-oxazolidine-2",4"-dione. This compound hasproved to be an extremely active anti-neoplastic agent but is alsodescribed in the aforesaid patent as being useful as anintermediate--see Col. 7 lines 49-58--for the preparation of thecorresponding 3"-(2-thioethyl) derivative. This latter compound isstated to be, in turn, useful as an intermediate in preparing thecorresponding N-(β-thioethyl)VLB 3-carboxamide (compound 25 in Conrad etal., loc. cit.).

It is an object of this invention to find improved methods forsynthesizing VLB 3"-(β-thioethyl)-3-spiro-5"-oxazolidine-2",4"-dione.

DESCRIPTION OF THE INVENTION

This invention provides an improved method of preparing compounds of theformula ##STR4## wherein one of R³ and R⁴ is OH or H and the other isethyl; R² is CH₃ or CHO; R¹ is OH or acetoxy and R is C₂ H₄ SR⁵ whereinR⁵ is H or CH₃. Also provided are new intermediates of the structure:

    Q--CH.sub.2 CH.sub.2 --S--S--CH.sub.2 --CH.sub.2 --Q--

    Q--CH.sub.2 --CH.sub.2 --S--S--CH.sub.2 --CH.sub.2 --NCO,

and

    Q--CH.sub.2 --CH.sub.2 --S--S--CH.sub.2 --CH.sub.2 --NH--CO.sub.2 alk

wherein alk is (C₁ -C₃)alkyl and Q is all of II except the R group atC-3" of the oxazolidinedione ring. Q can be represented by III below.##STR5## wherein R¹, R², R³ and R⁴ have the same meaning as hereinabove.

Also provided by this invention are new isocyanates of the formulaOCN--CH₂ --CH₂ --S--R⁶ wherein R⁶ is CH₃ or --S--CH₂ --CH₂ --NCO.

The 3"-thioalkyl oxazolidinediones represented by II above and by(Q--CH₂ CH₂ S--)₂ are prepared the following reaction: a Vinca dimer ofthe structure ##STR6## wherein R², R³ and R⁴ have their previouslyassigned meaning, is reacted with an isocyanate of the structureOCN--CH₂ --CH₂ --S--R to yield an oxazolidinedione according to formulaII above in which R is CH₂ --CH₂ --S--CH₃, or an intermediate accordingto III above in which R is CH₂ --CH₂ --S--S--CH₂ --CH₂ --N═C═O. Thislatter compound is not isolated as such ordinarily but as a reactionproduct with a lower alkanol --(C₁ -C₃)alkylOH--, having structure IIwherein R is CH₂ --CH₂ --S--S--CH₂ --CH₂ --NH--CO--O--(C₁ -C₃)alkyl andR¹ is acetoxy.

Reduction of the Vinca tetramer disulfide Q--CH₂ --CH₂ --S--S--CH₂ --CH₂--Q or of an intermediate Vinca dimer disulfide [III wherein R¹ isacetoxy and R is CH₂ --CH₂ --S--S--CH₂ --CH₂ --NCO or CH₂ --CH₂--S--S--CH₂ --CH₂ --NH--CO--O--(C₁ -C₃)alkyl], with zinc and acetic acidor other appropriate reducing agent yields the3"-(β-thioethyl)oxazolidine dione (III wherein R is CH₂ --CH₂ --SH).Oxidation of this thioethyl oxazolidinedione with ferrocycanide or othersuitable oxidizing agent having an E₀ of about +0.50 yields a Vincatetramer disulfide (Q--CH₂ CH₂ --S--)₂.

In the above reaction involving a Vinca dimer IV and an isocyanate, thesecondary hydroxyl at C-4 is acetylated. A 4-hydroxy if present, wouldreact with the isocyanate. It can be protected other than byacetylation, however.

It is believed that the formation of the oxazolidinedione ring startswith the reaction of the 3-hydroxy with an isocyanate to form acarbamate which, in a second step, cyclizes to an oxazolidinedione ringwith loss of CH₃ OH.

The 3"-thioalkyl oxazolidinediones represented by III when R is CH₂--CH₂ --SH or CH₂ --CH₂ --S--CH₃ and by (Q--CH₂ --CH₂ --S--)₂ are activeantimitotic and antineoplastic agents in their own right, but can alsobe converted by mild alkaline hydrolysis to carboxamide derivatives ofVLB, vincristine, leurosidine etc. and of the corresponding 4-desacetylcompounds (structure IV wherein The C-4 ##STR7## is replaced by a##STR8## and the corresponding disulfide--see Conrad et al., loc.cit--compounds 25, 26, 30 and 37 and U.S. Pat. No. 4,096,148, col. 7,line 32 et seq).

This invention is further illustrated by the following specificexamples.

EXAMPLE 1 Preparation of Vinblastine3"-(β-[β-(methoxycarbonylamino)ethyldithio]ethyl)-3-spiro-5"-oxazolidine-2",4"-dione

A solution was prepared from 40.4 g. of p-nitrophenylchloroformate and150 ml. of acetonitrile. This solution was added to a slurry of 22.5 g.of cystamine dihydrochloride and 150 ml. of anhydrous acetonitrile. Thereaction mixture was heated to refluxing temperature for about 2 daysafter which time it was cooled and filtered. The filter cake comprisingcystamine-bis-carbamate was crystallized from hot toluene. Thecrystalline product was then stirred with water for one-half hour toremove unreacted cystamine. This aqueous mixture was filtered and theprecipitate washed with water. Yield ofcystamine-bis(p-nitrophenyl)-carbamate was 19.5 g. melting at 163°-165°C.

nmr (DMSO-d₆): 2.91 (4H, t), 3.46 (4H, t), 3.82 (2H bis, exchangeablewith D₂ O), 7.41 (4H, d), 8.26 (4H, d) δ.

Infrared spectrum (mull); 3360, 1710, 1350, 1215 cm⁻¹.

Mass spectrum: m/e 204 (loss of carbamate from molecular ion plusfragmentation of the disulfide bond).

Analysis Calculated for: C₁₈ H₁₈ N₄ O₈ S₂ :

Theory: C, 44.81; H, 3.76; N, 11.61; S, 13.29.

Found: C, 45.05; H, 3.97; N, 11.80; S, 13.55.

Seven and nine-tenths grams of cystamine-bis(p-nitrophenyl)carbamate and2.9 g. of triethylamine were dissolved in toluene and the resultingsolution heated to 40° C. This solution was added in dropwise fashion toa solution of 3.1 g of trimethylsilylchloride in 25 ml. of toluene. Theresulting mixture was heated to 90° C. for about 3 hours, and was thencooled. Triethylamine hydrochloride precipitated and was separated byfiltration. The filtrate, containing cystamine-bis-isocyanate formed inthe above reaction, was concentrated to about 20 ml. 4.8 G. ofvinblastine free base were dissolved in the toluene solution containingthe isocyanate. This reaction mixture was heated to about 60° C. forabout 10 hours, followed by a heating period of 3 hours at about 80° C.Volatile constituents of the reaction mixture were then removed byevaporation in vacuo leaving 13.7 g. of an oil. This residual oil waschromatographed over 475 g. of silica gel (Woelm activity I). Thefollowing eluants were used, based on a 1:1 mixture ofmethylenedichloride and ethylacetate: 4 l. containing 5% methanol, onel. containing 7.5% methanol, 2 l. containing 10% methanol and 3 l.containing 15% methanol. Fractions shown by tlc to contain vinblastine3"-(β-[β-(methoxycarbonylamino)ethyldithio]ethyl)-3-spiro-5"-oxazolidine-2",4"-dionewere combined and the solvent removed from the combined products invacuo. Purified vinblastine3"-(β-[β-(methoxycarbonylamino)ethyldithio]-ethyl)-3-spiro-5"-oxazolidinedionethus prepared (1.24 g.) had the following physical characteristics:

Mass spectrum: m/e 895, 882, 865, 835, 804, 792, 649, 343, 525, 494,355, 154; field desorption 1014, 882.

nmr (CDCl₃): 0.68 (3H, t), 0.89 (3H, t), 2.02 (3H, s, NMe), 3.60 (3H, s,CO₂ Me), 3.66 (4H, CH₂ CH₂ S), 3.79 (3H, s, MeOAr), 5.26 (1H, m, C-6),5.41 (1H, s, C-4), 5.89 (1H, m, C-7), 6.11 (1H, s, C-17), 6.65 (1H, s,C-14), 7.2-7.5 (4H, m, Ar), 8.15 (1H, br s, NH) δ.

Infrared spectrum: 3600, 3460, 1814 (oxazolidinedionecarbonyl), 1745,1725, 1700 cm⁻¹.

cmr (CDCl₃): 154.6, 157.1, 169.8 δ (new carbonyls due tooxazolidinedione carbamate)

EXAMPLE 2 Preparation of Vinblastine3"-(β-thioethyl)-3-spiro-5"-oxazolidine-2",4"-dione

A solution was prepared from 507.1 mg. of vinblastine3"-(β-[β-(methoxycarbonylamino)ethyldithio]ethyl)-3-spiro-5"-oxazolidine-2",4"-dionesulfate and 20 ml. of glacial acetic acid. 317.7 Mg. of zinc dust wereadded and the resulting mixture heated to refluxing temperature forabout 1.5 hours. At this time, tlc showed that the reaction had goneessentially to completion. Ice was added to the reaction mixturefollowed by dilute aqueous ammonium hydroxide to pH=8. Water was addedand the resulting aqueous mixture extracted 6 times with an equal volumeof methylenedichloride. The methylenedichloride extracts were combinedand filtered and the solvent removed from the filtrate by evaporation invacuo. The resulting residue was dried with a toluene azeotrope. Yieldof vinblastine 3"-(β-thioethyl)-3-spiro-5"-oxazolidine-2",4"-dione thusprepared was 391.9 mg. The compound had the following characteristics:

nmr (CDCl₃): 0.67 (3H, t), 0.88 (3H, t), 2.01 (3H, s), 2.81 (m), 2.84(3H, s), 3.61 (3H, s), 3.68 (s), 3.79 (3H, s), 4.0 (m), 5.18 (1H, m),5.45 (1H, s), 5.88 (1H, s), 6.10 (1H, s), 6.64 (1H, s), about 7.5 (4H,m), 8.12 (1H, brs) δ.

Mass spectrum: m/e 895 (transmethylation), 881, 864, 850, 822, 820, 792,650, 543, 381, 354, 154, 135.

Infrared spectrum (CHCl₃): 3670, 3590, 3450, 1815, 1740, 1715, 1615cm⁻¹.

pK_(a) in 66% aqueous DMF=5.2, 7.0, 11.1.

The product was converted to the sulfate salt by standard procedures.

EXAMPLE 3 Preparation ofBis-[β-(vinblastine-3-spiro-5"-oxazolidine-2",4"-dione-3"-yl)ethyl]disulfide

A solution containing 327.6 mg. of vinblastine3"-(2-thioethyl)-3-spiro-5"-oxazolidine-2",4"-dione free base in about 2ml. of water was prepared by adding sufficient 12 N aqueous hydrochloricacid to solubilize the base. 375.1 Mg of potassium ferricyanide wereadded. There was an immediate heavy percipitate. The reaction mixturewas diluted with 30 ml. of water and the pH adjusted to about 8 by theaddition of 14 N aqueous ammonium hydroxide. The resulting aqueousmixture was extracted 5 times with an equal volume ofmethylenedichloride. The methylenedichloride extracts were combined. Thecombined extracts were concentrated by evaporation in vacuo and driedwith a toluene azeotrope. The residue containingbis-[β-vinblastine-3-spiro-5"-oxazolidine-2",4"-dione-3"-yl)ethyl]-disulfide(weight=331.7 mg.) was chromatographed over 35 g. of silica (Woelmactivity 1). The chromatogram was developed by employing 300 ml.fractions of a 1:1 ethylacetate-methylenedichloride solvent mixturecontaining increasing amounts of methanol from 4% up to 45%. Fractionsshown by tlc to contain the desired disulfide (R_(f) =0.25 in a 3:1ethylacetate-ethanol solvent mixture) were combined and the solventremoved: yield=55.9 mg. The compound had the following physicalproperties:

nmr (CDCl₃): 0.68 and 0.89 (12H, 2t), 2.03 (6H, s, OAc), 2.85 (6H, s,NMe), 5.08 (2H, m), 6.11 (2H, s), 6.65 (2H, s), 7.1-7.5 (4H, m, Ar),8.21 (2H, br s) δ.

Infrared spectrum (CHCl₃): 3680, 3600, 3460, 1815, 1745, 1720 (shoulder)cm⁻¹.

Mass spectrum: m/e 835 (cleavage of disulfide and loss of CH₂ S), 817,804, 775, 592, 494, 355, 154; field desorption 881.

Osmometric weight determination=2275 (theoretical 1760).

The sulfate salt was prepared by the usual procedure.

As previously stated, the compounds of this invention are antimitotics,and can adversely affect the growth of malignant cells. This activity ismanifested in a standard mitotic inhibition test employing Chinesehamster ovary cells.

Table 1 gives the results of this test for the compounds of thisinvention. In the Table, column 1 gives the name of the compound, column2 gives the dose in mcg/ml for accumulation in mitosis and column 3, thepercent mitotic accumulation as a range.

                  TABLE 1                                                         ______________________________________                                        Name of Compound         Dose   Range                                         ______________________________________                                        Vinblastine 3"-(β-(β-methoxycarbonyl-                               amino)ethyldithio]ethyl)-3-spiro-5"-                                          oxazolidine-2",4"-dione  20-2    7-10                                         vinblastine 3"-(β-thioethyl)-3-spiro-                                    5"-oxazolidine-2",4"-dione                                                                             20-2   10-15                                         ______________________________________                                    

The bases of this invention and their salts preparable by the processesdisclosed herein are active also in vivo against some but not alltransplanted tumors in mice. To demonstrate such activity, a protocolwas used involving the administration of the drug by the intraperitonealor oral route, at selected dose levels, against 6C₃ HED lymphosarcoma,B16 melanoma, 755 adenocarcinoma and C₃ H mammary carcinoma.

The following table--Table 2--gives the results of this experiment inwhich mice bearing the transplanted tumor were treated with a compoundof this invention. In the table, column 1 gives the name of thecompound; column 2, the name of the tumor; column 3, the dosage given;and column 4, the percent inhibition of tumor growth (I) or prolongationof life (PL). The following dosage regimen was employed: every fourthday (three doses) starting on the first day after inoculation.

                  TABLE 2                                                         ______________________________________                                                                           Percent                                                                       Inhibition                                                           Dose     or Pro-                                    Name of Compound Tumor    (mg/kg)  longation                                  ______________________________________                                        vinblastine 3"-(β-[β-(methoxy-                                                       6C.sub.3 HED                                                                           2.5      12*(I)                                     carbonylamino)ethyldithio]-                                                   ethyl)-3-spiro-oxazolidine                                                                     P1534(J) 2.5      16*(I)                                     2",4"-dione      P388/V   2.5      38(PL)                                                               2.0      25*(PL)                                                              1.5      30(PL)                                     vinblastine 3"-(β-thioethyl)-                                                             6C.sub.3 HED                                                                           2.5      14*(I)                                     3-spiro-5"-oxazolidine-                                                       2",4"-dione      P1534(J) 2.5      14*(I)                                                      P388/V   2.5      22*(PL)                                    bis-[β-(vinblastine 3-                                                                    6C.sub.3 HED                                                                           2.5      46(I)                                      spiro-5"-oxazolidone-2",4"-                                                   dione-3"-yl)ethyl]disulfide                                                                    P1534(J) 2.5      16*(I)                                                      P388/J   2.5      18*(PL)                                    4-desacetyl vinblastine 3"-                                                                    P388     1.4      77(PL)                                     (β-thioethyl)-3-spiro-5"-                                                oxazolidine-2",4"-dione   1.2      65(PL)                                                               1.0      52(PL)                                     ______________________________________                                         In the above Table, 6C.sub.3 HED is a lymphosarcoma and P1534(J) and          P388/J are leukemias.                                                         *Percentages below 30 are not statistically significant.                 

In utilizing the novel compounds of this invention as anti-tumor agentsin mammals, either the parenteral or oral route of administration may beemployed. For such use, the drug is customarily mixed with apharmaceutically suitable carrier. With parenteral administration, theintravenous route is preferred although, with smaller mammals such asmice, the intraperitoneal route may be used. For intravenousadministration, isotonic solutions containing about 1 mg./ml. of a saltof an alkaloidal base of formula II above are employed. The drug isadministered at a dose of from 0.01 to 10 mg./kg. of mammalian bodyweight once or twice a week or every two weeks depending on both theactivity and the toxicity of the drug. An alternative method of arrivingat a therapeutic dose is based on body surface area with a dose in therange 0.1 to 10 mg./meter squared of mammalian body surface administeredevery 7 or 17 days or twice weekly.

As would be expected, the compounds of this invention differ in theiranti-tumor spectrum from that of VLB, vincristine and vindesine in thesame way that the anti-tumor spectra of those compounds differ amongthemselves, one drug being more effective against certain tumors orclasses of tumors and less effective against others. However, inutilizing the compounds of this invention clinically, an oncologistwould administer one of them initially by the same route in the samevehicle and against the same types of tumors as employed clinically withvindesine, vincristine and VLB. Differences in dosage level would, ofcourse, be based on relative oncolytic potency and toxicity.

We claim:
 1. A compound of the formula

    Q--CH.sub.2 --CH.sub.2 --S--S--CH.sub.2 --CH.sub.2 --Z

wherein Q is a 3"-oxazolidindionyl radical of the formula: ##STR9##wherein R² is CHO or CH₃ ; one of R³ and R⁴ OH or H and the other isethyl; and Z is Q, NCO or NH--CO₂ --(C₁ -C₃)alkyl.
 2. Anoxazolidinedione of the formula: ##STR10## wherein one of R³ and R⁴ isOH or H and the other is ethyl; R² is CH₃ or CHO; R¹ is OH or acetoxyand R is CH₁ --CH₂ --S--S--CH₂ --CH₂ --R⁹ wherein R⁹ is NCO orNH--CO--O--(C₁ -C₃)alkyl.
 3. A process for preparing a compound of thestructure

    Q--CH.sub.2 --CH.sub.2 --SH

wherein Q is a 3"-oxazolidinedioxyl radical of the formula ##STR11##wherein R¹ is OH or acetoxy; R² is CH₃ or CHO; and one of R³ and R⁴ is Hor OH and the other is ethyl, which comprises reducing a compound of theformula Q--CH₂ --CH₂ --S--S--CH₂ --CH₂ --Z, wherein Z is Q, NCO orNH--CO₂ --(C₁ -C₃)alkyl; and wherein R¹ is acetoxy with zinc and aceticacid; and then, to produce a compound in which R¹ is OH, subjecting thereduced compound to mild alkaline hydrolysis.
 4. The process whichcomprises oxidizing a compound of the structure Q--CH₂ --CH₂ --S--Hwherein Q is a 3"-oxazolidinedionyl radical of the formula ##STR12##wherein R² is CH₃ or CHO; R¹ is OH or acetoxy and one of R³ and R⁴ is Hor OH and the other is ethyl;with ferricyanide in hydrochloric acid. 5.A compound according to claim 1, said compound being vinblastine3"-(β-[β-(methoxycarbonylamino)-ethyldithio]ethyl)-3-spiro-5"-oxazolidine-2",4"-dione.6. A compound according to claim 1, said compound being vinblastine3"-(β-[β-isocyanatoethyldithio]ethyl)-3-spiro-5"-oxazolidine-2",4"-dione.